The gram-positive bacterium Staphylococcus aureus is a common human pathogen causing food poisoning, skin infections, abscesses, bacteraemia, pneumonia, endocarditis, osteomyelitis, toxic shock syndrome, staphylococcal scarlet fever and scalded skin syndrome. S. aureus produces a plethora of cell-surface and secreted proteins that bind host cell-surface receptors, extracellular matrix proteins, and soluble serum factors involved in both innate and adaptive immunity (1). Several studies (2) and the recent completion of several staphylococcal genomes has revealed that many of these virulence factors are clustered within 3 distinct pathogenicity islands SaPIn1, SaPIn2 and SaPIn3 (3, 4). One important class of genes within SaPIn1 and SaPIn3 are those that code for superantigens. These potent exotoxins target the antigen recognition receptors of the adaptive immune response—the Major Histocompatibility Complex class II (MHC-II) and T cell Receptor (TcR) and drive antigen independent T-cell activation and cytokine release (5) possibly in an attempt to prevent local inflammation and leukocyte recruitment to the site of infection through the production of T cell cytokines (4).
The staphylococcal exotoxin-like proteins (SETs) encoded by genes clustered within the staphylococcal pathogenicity island SaPIn2 are superantigen homologues (8). Comparative alignment of SETs and superantigen sequences indicate that they have evolved from the same ancestral gene (3) and are most similar to toxic shock syndrome toxin 1 (TSST-1) whose gene resides on SaPIn1. TSST-1 is a TcR Vβ2 binding superantigen produced by toxigenic strains of S. aureus associated with Toxic Shock Syndrome (9). Twenty-six members of the SET family have been identified (8) (3) (10), although several appear to be allelic variants. For example SET1 from strain NCTC6571 (8), SET11 from N315 and Mu50 (3), and SET22 from MW2 (10) are probably the same protein. The 3-D structure of SET3 has been determined and displays the same OB-fold/β-grasp two-domain structure as the superantigen (11) with conservation of the central core region at the interface of the two domains. Yet the SETs are not superantigens. They do not bind MHC class II nor activate T cells. Those regions on SET3 corresponding to the MHC class II or TcR binding sites on superantigens are substantially altered (11). The function of SETs remains unknown although the presence of their genes on SaPIn2 may indicate that they are part of the bacterial defense armamentarium (11) (8) (12). Notably an set15− mutant of S. aureus displayed a 30-fold reduction in bacterial persistence in a murine kidney abscess infection model.
SET proteins may also be known as staphylococcal superantigen-like proteins (or SSLs). There has been a move recently to use SSL when naming these proteins.
IgA is the predominant antibody located at mucosal surfaces and the second most predominant isotype in serum. In humans the IgA production of approximately 66 mg/kg/day exceeds that of all other Ig classes combined (13). Mucosal IgA exists mostly as a dimer complexed with a J-chain and a secretory component (SC). Polymeric IgA binds to the polyIg receptor on the basolateral surface of the mucosal epithelium to be transcytosed through the epithelial layer and released into the mucosa as a covalent complex called secretory IgA (sIgA). The cleaved ectodomain of the pIgR remains bound as the secretory component (SC). Secretory IgA is unable to bind FcαR and activate phagocytosis in the absence of an integrin co-factor Mac-1 (complement receptor 3 or CD11b) (17). (Reviewed in (15), (16)). Serum IgA on the other hand is predominantly monomeric and only binds avidly to its receptor FcαRI (CD89) on formation of immune complexes. Binding and cross-linking of FcαRI (CD89) by IgA immune complexes initiates phagocytosis by neutrophils, granulocytes and monocyte/macrophages (17). Monomeric serum IgA has been proposed to provide a second line of defense against microbes such as S. aureus via FcαRI mediated phagocytosis (18, 19). Mutagenesis (20-22) and the recent crystal structure of an IgA:FcαR complex reveals that the FcαR Ig-like D1 domain binds at the Cα2:Cα3 junction of the IgA H-chain (15).
Complement C5 is the central component in the terminal stage of the classical, alternative, and lectin mediated complement pathways. Complement C5 is ˜189 kD and is synthesised as an intracellular single-chain precursor that is secreted as a two-chain glycoprotein consisting of a 75 kD N-terminal C5β fragment disulfide linked to a 115 kD C-terminal C5α fragment ((23, 24)). The surface bound C5 convertases generated from either the classical, alternative or lectin pathway; cleave soluble C5 to generate two active fragments C5a and C5b. The potent anaphylatoxin C5a is a 74-residue N-terminus fragment cleaved from C5α by C5 convertase. C5a binds a G-protein coupled receptor C5aR on the surface of myeloid cells to stimulate a range of pro-inflammatory and chemotactic actions such as oxidative burst, phagocytosis and leukocyte recruitment which all contribute to the defense against organisms such as S. aureus (25). The C5b fragment initiates assembly of the terminal complement components into the membrane attack complex (MAC) that forms a water permeable membrane channel leading to cell lysis.
Methods are known for the purification of immunoglobulins, including IgA. For example, purification may be achieved by methods including classical protein purification techniques, such as ion exchange and size exclusion chromatography, and by affinity purification using a number of bacterial derived proteins, such as Protein L. These techniques may suffer from a number of problems including being laborious, lacking specificity and can result in low yields and purity. For example, Protein L, derived from the bacterium Peptostreptococcus magnus, and which is used for purification of immunoglobulins, binds to a wide variety of immunoglobulins. This means that it may not be suitable for the purification of a single species of immunoglobulin from a heterogenous mix of immunoglobulins.
An example of a product which may allow for single step purification of IgA and IgE is the affinity purification product known as Kaptiv-ae™ (Genomics One International Inc, USA). This is a synthetic peptidomimetic compound that selectively binds IgA and IgE from several species.
Single step purification methods for complement C5 are not known. Current methods to purify complement C5 from human serum, for example, rely on multiple chromatographic steps such as ion exchange and size exclusion chromatography. These may often result in low yields of final product.
Accordingly, there may be considered a need to provide an alternative or improved method of isolating and identifying IgA and C5.
Bibliographic details of the publications referred to herein are collected at the end of the description.